---+ UV mutagenesis of Bacteria ---++ Determination of Optimal UV treatment This procedure is used to determine optimal treatment which will be used for library generation. 1 Grow culture overnight as appropriate for strain. 1 Pellet 1mL of overnight culture at 3k rcf for 5 minutes. * see note 1 1 Aspirate media. 1 Resuspend in 1mL sterile saline. 1 Label sterile petri dishes for each of the for each condition to be tested for each strain. * see note 2 * Typical conditions (all in µJ/cm<sup>2</sup>): * 0 -- required for determining death rate! * 5,000 * 10,000 * 15,000 * 20,000 * 25,000 * 30,000 1 Near the UV crosslinker to minimize contamination concerns, transfer 120µl of cells to the center of each petri dish. Cells should form a distinct droplet. 1 Place the dish in the UV crosslinker for the appropriate treatment. Essential to remove the lid from the petri dish. * good practice to place the 0 sample in the crosslinker as well. 1 Transfer 100µL from the droplet to a labeled tube some careful pipetting can increase number of cells recovered as cells may settle during experiment. Be careful not to introduce air bubbles. 1 Plate for ~200 cells assuming 0 death rate. * replicate plating or higher number of cells can be used to increase confidence in death rate. 1 Grow o/n 1 Calculate death rate based on number of colonies. * see note 3 1 Update following table for future reference. ---++ Table of previously determined optimal (~95-99% death ratios). Please update with additional results. |*Strain ID*|* µJ/cm<sup>2</sup>*|*Strain description*|*Researcher*| |SKO16|27,500|BWA25113 with plasmid|DED -- via SKO| |unk|10,000|acenobacter|BR| ---++ Library Generation 1 Grow culture overnight as appropriate for strain. 1 Pellet 1mL of overnight culture at 3k rcf for 5 minutes in each of 4 eppendorf tubes. 1 Aspirate media. 1 Resuspend each in 1mL sterile saline. 1 Label 2 sterile petri dishes with 0 and optimal µJ/cm<sup>2</sup> treatment for each strain. 1 Near the UV crosslinker to minimize contamination concerns, transfer a total of at least 11 120µl droplets of cells distributed around the dish. Typically up to 6 droplets can easily be kept distinct on a single plate. 1 Place each dish in the UV crosslinker for the appropriate treatment. Essential to remove the lid from the petri dish. * good practice to place the 0 sample in the crosslinker as well. 1 Transfer 100µL from each droplet to a labeled tube. Some careful pipetting up and down can lead to recovery of more cells as they may settle during experiment. Take care to not introduce air bubbles. 1 Pellet cells at 3k rcf for 5 minutes. 1 Resuspend in appropriate growth media. 1 Transfer appropriate amount of cells to o/n culture. This can either be a dilution or nearly the entire volume of cells depending on how large of a library you want to start with. 1 Plate both treated and control for ~200 cells assuming 0 death rate to estimate total number of viable mutants. * replicate plating or higher number of cells can be used to increase confidence in death rate. 1 Grow plate and culture o/n. 1 Calculate number of mutants based on number of colonies. * This is expected to be very similar to previously calculated death ratios x the total number of cells treated. 1 Freezedowns should be created for o/n culture of viable cells and possibly resuspended treated cells depending on downstream applications. ---+++ Notes 1 Single attempt at using 1/3 of the total concentration of cells did not result in any noticable differences. DED SKO GFP project. 1 Conceivably 1 petri dish could be used with multiple strains, but risk of contamination obviously higher. * _This has not been attempted to my knowledge (remove if you attempt with confident results or warning to not attempt in future)._ 1 Not suggested to extrapolate kill ratios, best to test additional conditions if 95<sup>+</sup>% death rate not achieved. -- Main.DanielDeatherage - 13 Feb 2014
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Topic revision: r1 - 2014-02-13 - DanielDeatherage